October 1, 2015;
A complex network of transcription factors regulates specification of neural crest cells at early neurula
stage by stabilizing neural crest identity and activating neural crest effector genes so that distinct subpopulations evolve. In this network, c-myc
acts on top of the gene hierarchy controlling snail2
) expression. While snail2
are well studied neural crest specifier genes little is known about the role of phb1
in this process. To identify phb1
regulated genes we analyzed the transcriptome of neural crest explants of phb1
morphant Xenopus embryos. Among 147 phb1
regulated genes we identified the membrane-associated protein-tyrosine phosphatase PRP4A3 (prl3
) and the atypical cadherin and Wnt-PCP component van gogh like1 (vangl1
). Gain of function, loss of function and epistasis experiments allowed us to allocate both genes in the neural crest specification network between phb1
. Interestingly, both, vangl1
regulate only a small subset of neural crest marker genes. The identification of two membrane-associated proteins as novel neural crest specifiers indicates that in addition to gene regulation by combinatory effects of transcription factors also post-translational modifications (prl3
) and cell-cell adhesion and/or regulation of cell-polarity (vangl1
) specify the identity of neural crest cell populations. genesis 53:627-639, 2015. © 2015 Wiley Periodicals, Inc.
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Figure 1. Identification of phb1 target genes in the neural crest. (a) Neural crest explants of 300 Xenopus neurula embryos were pooled to gain samples of total RNA. The quality of total RNA was checked by calculating the RIN-value. Pools with a RIN-value > 8 were analyzed on Affimetrix GeneChip. Each experiment was performed in triplicates. To extract putative phb1 regulated genes among the roughly 40,000 spots on the GeneChip we selected those candidates which were downregulated more than two-fold in the phb1 morphants (P < 0.2) and back-regulated more than 1.5 fold (P <1.5) in the morphants co-injected with phb1 mRNA. The complete list of these 147 putative phb1 targets is found in Supporting Information Table 2. (b) The indicated morpholinos were injected together with dextran-TRITC (dex) in one animal dorsal blastomere of 8 cell stage embryos. The injected side is marked with asterisks. Embryos were fixed at stage 18 and analyzed for the expression of twist. The reduction of twist expression (white arrows) correlates with the morpholino doses. Scale bar = 400 µm. (c) and (d) Quantification of the twist expression illustrates the dose dependence and significance of reduction in the morphants. ***P < 0.005, *P < 0.05, n = number of embryos.
Figure 2. Vangl1 cannot be replaced by phb1 and prl3 and prl3 cannot be replaced by vangl1. (a) The vangl1 specific antisense morpholino was co-injected with the indicated rescue constructs in one animal dorsal blastomere of 8 cell stage embryos. Embryos were fixed at stage 18 and analyzed for the expression of twist. Twist expression in the morphants was restored by vangl1-rescue mRNA but not by prl3 or phb1 mRNA. (b) shows the quantification of the results. ***P < 0.005, **P < 0.01, ns = not significant, n = number of embryos. (c) The prl3 specific antisense morpholino was co-injected with the indicated rescue constructs in one animal dorsal blastomere of 8-cell stage embryos. Embryos were fixed at stage 18 and analyzed for the expression of twist. Twist expression in the morphants was restored by 100 pg prl3-rescue mRNA and by phb1 mRNA but only slightly by vangl1 mRNA. (d) Shows the quantification of the results. Importantly, restored expression of twist was only observed by low doses (100 pg) of prl3 rescue mRNA, but not by high doses (500 pg) ***P < 0.005, *P < 0.05, ns = not significant, n = number of embryos.
Figure 3. Overexpressed vangl1 upregulates twist expression, overexpressed prl3 represses twist expression. Embryos were injected in 8-cell stage in one animal dorsal blastomere with indicated amounts of mRNA. In situ hybridization was performed at stage 18 with twist probe. Asterisks mark injected side. (a) Overexpression of 500 pg vangl1 mRNA results in a clear upregulation of twist expression. (b) Statistics revealed high significance of twist upregulation. 47% of the morphants showed enhanced twist expression. (c) Overexpression of prl3 results in a downregulation of twist expression. (d) Statistics show that twist reduction is significant even at very low doses (50 pg). ***P < 0.005, **P < 0.0, n = number of embryos. Scale bar = 400 µm.
Figure 4. Vangl1 and prl3 regulate only a small subset of neural crest specific marker genes. (a) Prl3 and vangl1 morphants were analyzed at stage 18 for the expression of the neural crest specific marker genes foxD3, sox2, AP-2, snail1, phb1, and snail2. (b) Shows quantification of the results in vangl1 morphants and (c) shows quantification of prl3 morphants. ***P < 0.005, n = number of embryos.
Figure 5. Vangl1 and prl3 coinjection can restore twist expression in phb1 morphants. 16 ng phb1 specific antisense morpholino were co-injected with 500 pg of the indicated mRNA and dextran-TRITC (dex) in one animal dorsal blastomere of 8-cell stage embryos. (a) Twist expression was analyzed in stage 18 embryos. Asterisks mark the injected side. The reduction of twist expression was quantified in (b). ***P < 0.005, n = number of embryos.
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